Luciferase is an enzyme derived from a luminescent bacterium. The enzyme catalyzes a reaction that involves a reduced flavin mononucleotide (hereinafter referred to as "FMNH.sub.2 ") and a long chain fatty aldehyde as substrates in the presence of oxygen. An oxidized flavin mononucleotide (hereinafter referred to "FMN") and a long chain fatty acid are produced thereby along with the emission of a blue color light.
In the cell, the substrate FMNH.sub.2 used in this reaction is supplied by the reducing action of nicotinamide adenine dinucleotide:flavin mononucleotide (NADH:FMN) reductase and nicotinamide adenine dinucleotide phosphate:flavin mononucleotide (NADPH:FMN) reductase; and the long chain fatty aldehyde is supplied from a fatty acid reductase complex.
Recently, Spyrou et al. (J. Bacteriol., 173, 3673-3679 (1991)) isolated a flavin reductase gene from Escherichia coli and elucidated its primary structure.
The present inventors have also isolated a FMN reductase gene, that of the luminescent bacterium, Vibrio fischeri. The inventors have determined its nucleotide sequence, and further have succeeded in expressing the gene in E. coli (Japanese Patent Application No. Hei 03-351,717). However, isolation of the flavin reductase gene from the luminescent bacterium Xenorhabdus luminescens and its expression in E. coli has not yet been reported.
FMNH.sub.2 is easily and instantaneously autoxidized in air and converted into FMN. In order to have the luminescent ability of bacterial luciferase displayed to the maximum, it is necessary to continuously supply the FMNH.sub.2 as a substrate. FMN reductase is most important in order to achieve this object.
As described above, this enzyme conjugates with the bacterial luciferase and catalyzes the reaction of converting FMN as the product of the luciferase reaction into FMNH.sub.2 ; thus, luciferase and FMN reductase are made coexistent in the reaction system; and as a result, it is possible to retain the luminescent reaction. Namely, since the bacterial luciferase is many times subjected to turnover, the bacterium retains the luminescence as far as the long chain aldehyde in a large excess is existent in the reaction system.
As described above, FMN reductase is indispensable for making the most of the bacterial luciferase, and by obtaining the gene, it is possible to prepare FMN reductase enzyme in large quantities.
In view of this, it is an object of the present invention to provide nucleic acids coding for flavin reductase of a luminescent bacterium, X. luminescens. It is another object of the invention to provide vectors comprising such nucleic acids, and host cells into which these vectors have been introduced. Yet another object of the invention is to provide recombinant methods for the production of substantially purified recombinant flavin reductase, wherein the protein is expressed in a host cell of the present invention. A further object of the invention is to provide recombinant flavin reductase produced in accordance with such methods.
These and other objects and advantages of the invention, as well as additional inventive features, will be more readily apparent from the description of the invention provided herein.